Heat shock proteins (hsps, HSPs) are a family of highly conserved proteins that are widely distributed throughout the plant and animal kingdoms. On the basis of their molecular weight, the major heat shock proteins are grouped into six different families: small (hsp20-30 kDa); hsp40; hsp60; hsp70; hsp90; and hsp100. Heat shock proteins are ubiquitously expressed in both prokaryotic and eukaryotic cells, where they function as chaperones in the folding and unfolding of polypeptides. A further role of heat shock proteins is to chaperone peptides from one cellular compartment to another and, in the case of diseased cells, heat shock proteins are also known to chaperone viral or tumour-associated peptides to the cell-surface for presentation to the immune system.
Although heat shock proteins were originally identified in cells subjected to heat stress, their production can result from a number of other forms of stress, such as infection, osmotic stress, cytokine stress and the like. Accordingly, heat shock proteins are also commonly referred to as stress proteins (SPs) on the basis that their expression is not solely caused by a heat stress. However, transcriptional analysis of the genes induced by different stress stimuli show the induction of distinct sets of genes following exposure of a cell to different stress inducing stimuli (Bacon & Marsh (2007) Curr. Mol. Med. 7:277-86). Moreover, the individual stress regulons are independently induced (vanBogelen et al. (1987) J. Bact. 169:26-32 and Wilkes et al. (2009) Applied and Environmental MicrobioL 75:981-990) and regulated (Holmes et al. (2010) Microbiology 156:158-166). In particular, vanBogelen shows that different stress inducing stimuli cause distinct heat shock protein genes to be expressed and that the specific expression of these genes resulted only from one type of stress inducing stimulus when four stress inducing stimuli were tested.
While heat shock proteins can themselves be used as the immunogenic determinant in vaccine compositions, it has been observed that complexes formed between heat shock proteins and peptides, in particular antigenic peptide fragments, mediate an enhanced immune response when administered to a subject. Furthermore, it is known that heat shock proteins can produce complexes which can be classed as either constitutively produced heat shock protein complexes or induced heat shock protein complexes. Constitutively produced heat shock protein complexes are those comprising heat shock proteins which are produced under normal homeostatic conditions. Induced heat shock protein complexes are produced when a cell is exposed to conditions of stress. When in a stressed state, the cell upregulates the production of stress proteins, with these upregulated heat shock proteins being known as induced heat shock proteins. Furthermore, these induced heat shock proteins form complexes with peptide fragments which are seen to be more immunogenic than complexes formed when a cell is not under conditions of stress. The enhanced immunogenicity of such induced heat shock protein complexes over constitutive heat shock protein complexes has been exemplified in WO 01/13943. Without wishing to be bound by theory, the inventors predict that the enhanced immunogenicity observed with induced heat shock protein complexes is not due to the actual stress protein component of the complex being different, but due to the stress conditions causing proteins within the cell to unfold or denature. The heat shock proteins therefore complex with proteins or protein fragments to prevent them unfolding, or to refold them, as well as complexing with protein fragments as part of the cell's antigen processing pathway.
Vaccine compositions which comprise heat shock protein complexes (which may also be referred to as stress protein complexes) as the immunogenic determinant are widely known. Such vaccines have significant potential as they show the promise of conferring broad, protective immunity against infection and disease. It has also been widely documented that heat shock protein complexes are efficacious as vaccines against specific cancers. It has been shown that pathogen derived stress protein complexes isolated from heat-shocked BCG cells induced T-helper 1 (Th1) lymphocyte mediated immune responses in a vaccinated host, which conferred protective immunity against a live challenge in a murine aerosol challenge model of pulmonary tuberculosis (International PCT Patent Application No. WO 01/13944). Moreover, it has been shown in WO 02/20045, WO 00/10597 and WO 01/13943 that stress protein complexes isolated from pathogens or pathogen infected cells are effective as the immunogenic determinant within vaccines against infectious diseases.
There have been various approaches to producing vaccine compositions comprising stress protein complexes for the treatment and prevention of infectious diseases. WO 95/24923 discloses constitutive heat shock protein complexes comprising a heat shock protein derived from a host eukaryotic cell and an antigenic peptide fragment derived from a pathogen. WO 01/13943 discloses heat shock protein complexes which are induced following the use of a stress inducing stimulus such as heat or a cytokine such as tumour necrosis factor (TNF). Said stress proteins may comprise stress proteins derived from a host cell, or, alternatively, stress proteins derived from an invading pathogen, said stress proteins being complexed to a peptide derived from the invading pathogen. WO 01/13944 discloses stress protein complexes which are produced following a pathogen being subjected to a stress inducing stimulus, wherein the stress protein and associated peptide fragment are derived directly from the pathogen.